The invention relates to novel genetic markers for wheats of the species Triticum aestivum and closely related species of the tribe Triticeae and to the use of said markers.
The most widely spread, known, DNA-based genetic markers are the so-called restriction fragment length polymorphisms (RFLP) markers. For using these markers, genomic DNA is digested with restriction enzymes, separated on agarose gels and transferred to nylon membranes (Southern Blot). Specific fragments are detected by hybridization with radioactively labeled DNA probes. When mutations occur in the region of the restriction enzymes used or when smaller deletions/insertions occur, polymorphisms between different lines are found, which are passed on stably and mostly codominantly. The use of RFLP markers in hexaploid cultivated wheat is possible only to a limited extent, since only very little polymorphism is detected in wheat in this manner.
It has already been described that microsatellite markers detect significantly more polymorphism between different wheat lines than do RFLP markers. This can be attributed particularly to the occurrence of multiple alleles per locus (Rbder et al., Mol. Gen. Genet. (1995) 246, 327-333). Moreover, it is known that microsatellite markers have the advantage that they can be detected by way of PCR and that therefore large amounts of samples can be analyzed more easily.
It is an object of the invention to provide novel microsatellite markers for the genetic analysis of plants of the Triticum aestivum species, which markers are distinguished by a degree of DNA polymorphism, which is higher than that of other molecular probes, that have been developed previously for the wheat genome.
This objective is accomplished as follows. The inventive markers are based on the amplification of certain hypervariable genome sections, the so-called microsatellites, with the help of their polymerase chain reaction (PCR). For specific amplification, two primers, in each to the case left and the right in the flanking sequences, are required for each microsatellite locus. On the average, these primers are 20 i 3 bases long and are defined by their sequences. In principle, a microsatellite marker is a sequence tagged site (STS), which is defined by two specific primers. These primers flank, in each case to the left and the right, a so-called microsatellite sequence. A microsatellite sequence is defined as a tandem repetitive repetition of a di-, tri- or tetranucleotide sequence, for example (GA)n, in which n greater than 10. Composite microsatellite sequences also occur, such as (GT)n(AT)n, as well as imperfect sequences, in which individual bases are mutated, such as (GA)nCA(GA)n. Among various lines and varieties, there is variation in the number of repeats at a certain locus. After amplification of the microsatellites, this leads, by means of the specific primers in the flanking sequences, to PCR products of different length and, with that, to polymorphisms. These polymorphisms are passed on stably and can therefore be used as genetic markers. In some cases, null alleles (no visible fragment) also occur, when there are mutations within the binding site for the primers.
The separation and detection of the PCR products obtained can be carried out with different technical variants. For separating the fragments, highly resolving agarose gels, native polyacrylamide gels or denaturing polyacrylamide gels (=sequencing gels) can be used. Depending on the separation system, fragments are detected using ethidium bromide staining, silver staining or, after labeling the PCR fragments radioactively, using autoradiography. A further, very effective variation for separation and detection consists of the use of an automatic sequencer with dye- or fluorescence-labeled primers. For this purpose, it is necessary to synthesize a dye- or fluorescence-labeled primer from each microsatellite primer pair. PCR amplification results in a labeled product, which can be detected by the sequencing equipment. At the same time, dye- or fluorescence-labeled size standards are also separated for each sample in the same track. After that, special software enable the absolute size of each fragment, which has been separated, to be calculated and, with that, also permits fragments from different gel runs to be compared. With this method, several hundred samples can be analyzed largely automatically in a day.
Pursuant to the invention, microsatellite markers are made available, which contain the following primer pairs with assigned microsatellite sequences or a number thereof and amplify the loci of all chromosomes of the wheat genome and therefore find use for gene marking.
These markers are distinguished by a high degree of polymorphism between different wheat varieties or lines and usually detect several alleles per genetic locus in different wheat lines.
They can therefore be used for DNA fingerprinting, species identification, relationship or similarity studies, characterization of cytological lines, such as deletion lines, substitution lines, addition lines, etc. and all forms of genetic mapping, including mapping of individual genes and quantitative trait analysis features (QTLs). In addition, their use is also very suitable for automation and it is possible to carry out the detection of the products with nonradioactive methods.
With the help of these inventive markers the possibility is provided, for example, of differentiating almost all European wheat lines.